Modification of culture medium for Xenopus embryonic cells

Printed in Sweden Copyright 0 1974 by Academic Press, Inc. Al! rights of wproduction in any form resewed

Experimental Cell Research 88 (1974) 24 l-246

MODIFICATION

OF CULTURE

MEDIUM

FOR XENOPUS

EMBRYONIC

CELLS 1. Effects of Albumin and Other Proteins T. NAKAHASHI Department

and K. YAMANA

of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan

SUMMARY Stearns medium that had originally been developed for culture of Ram pipiens embryonic cells was modified and used for Xenopus luevis embryonic cells. Modification was done either by reducing a concentration of albumin or by replacing it with other proteins, such as cr-globulin, B,y-globulin, horse serum and protamin. Comparisons were made between the original and the modified medium with regard to their ability to support not only cellular uptake of 3H-uridine, 3H-thymidine and W-protein hydrolysate, but also aggregate formation. From the results obtained it has been shown that the original Stearns medium containing 0.5 % albumin and the modified medium, which includes 0.1 % globulin in place of albumin, seem to be most suited also for Xenopus embryonic cells. A concentration of albumin can be reduced to 0.1 % without lowering the rate of 3H-uridine incorporation or RNA synthesis, but with an apparent decrease in the 3H-thymidine and X-protein hydrolysate incorporation. In a medium containing cr-globulin the cells are more active to incorporate 3H-thymidine and W-protein hydrolysate than in media containing B,y-globulin and horse serum as well as albumin. No remarkable difference is found among the media tested in their ability to support aggregate formation and in the appearance of aggregates formed. In a medium with protamin there occurs neither incorporation of the precursors nor formation of aggregates.

work [l, 21 from this laboratory has shown that isolated cells derived from blastulae and neurulae of Xenopus laevis incorporate a variety of radioactive precursors very actively into their nucleic acid and protein. As amphibian embryos are not especially permeable to exogenous substances, our method [l, 21 and its modification [3] should provide an opportunity for biochemical investigations on early embryogenesis. In these works [l-3] Stearns medium [4] was used for culture of isolated embryonic cells. However, it was originally developed for embryonic cells of Rana pipiens and consists of a balanced salt solution and 0.5 % Previous

albumin. The composition of a culture medium, especially the serum and protein contained, has long been known to have a remarkable effect on the morphology and also on the biochemical activities of the cells, though their role remains to be defined [5]. The present report compares Stearns medium with its modification (which includes various proteins instead of albumin) in order to find out whether Stearns medium is suited also for Xenopus embryonic cells and, if not, to find a starting point for improvement. The criterion for ‘suitability’ of a medium is the proclivity of the cells to incorporate radioactive labels of nucleic acid and protein Exptl Cell Res 88 (1974)

242

Nakahashi and Yamana

and to form aggregates during culture. These activities are likely to reflect their normal activity and behavior in the embryo and, furthermore, are of primary concern in our present and future investigations. It has been shown that media containing albumin or a-globulin are the most suited also for the culture of Xenopus embryonic cells, and the other proteins support the cellular activity and aggregate formation to lesser extents.

A set of experiments was designed to compare at one time two or three of the modified media with the original (or control) medium, using a single batch of embryos. Each set of experiments was repeated 3 to 5 times with embryos of different batches. Although the values obtained varied from batch to batch, the essential feature was completely consistent and uniform. A direct comparison was made between the results obtained with a single batch of embryos.

RESULTS AND DISCUSSION Determination of UV absorption and phosphorus content of acid-soluble fractions

MATERIALS

AND METHODS

Embryos of Xenopus laevis were obtained and dissociated as previously described [I, 21. Cells prepared from 10 blastulae (stage 8) [6] were cultured and labeled in 2 ml of the media to be tested in a glass Petri dish (0 3.5 cm). The cells were withdrawn after 4, 10 and 24 h of culture. After being labeled the cells were homogenized, and TCA-soluble and insoluble fractions were prepared. Hot TCA hydrolysate was also obtained from the latter. These were dried and the radioactivity was measured as previously described [I, 21. RNA was extracted from the homogenate with phenol-SDS-bentonite [I, 21, and fractionated into M NaCl-soluble and insoluble fractions: these corresponded roughly to transfer and 5S RNAs, and 18s and 28s RNAs, respectively. The radioactivity was assayed in a scintillation spectrometer as previously described [7]. Acid-soluble fractions were prepared from the cells that had been cultured for 24 h in media. They were applied onto small columns of charcoal, which were then eluted with ammoniac ethanol. The UV absorption and phosphorus content were measured following conventional methods. The DNA content was also determined in the acid-insoluble fractions. The culture medium used was that of Stearns & Kostellow [4], except that either a concentration of albumin (bovine plasma, fraction V: Armour Pharmaceutical Co.) was reduced, or albumin was replaced with the following: a-globulin (0.1 %: human serum, fraction lV-4: National Biochemical Corp.), P,y-globulin (0.1 %, bovine serum, fraction II +III; Miles Laboratory, Inc.), protamine sulphate (0.1 % or 0.5 %; Yuki Gosei Kogyo) and horse serum (3 56, TC-horse serum; Chiba Kessei). In some experiments they were dialysed before use. The following labeled precursors were used: 3Huridine-5T (I &i/ml; 5.0 Ci/mmole), 3H-thymidine-6T (1 &i/ml; 0.5 Ci/mmole) and W-protein hydrolysate (0.05 ,&i/ml; 1.99 mCi/mmole). In some cases, the specific radioactivity of 3H-uridine was lowered to 0.5 Ci/mmole. These precursors wer-eall purchased from Daiichi Pure Chemicals Co. The aggregates formed were examined after a 24 hculture period with a microscope: their number was counted and their size measured. Exprl Cell Res 88 (1974)

In the present experiments, we have attempted to obtain the relative, not absolute, rate of RNA synthesis. The incorporation into RNA and acid-soluble fractions and the content of acid-soluble ribonucleoside derivatives were measured. Since ribonucleoside triphosphates were the major nucleoside derivatives in the acid-soluble fraction of Xenopus eggs [8], the UV absorption and organic phosphorus content were determined and taken as rough measures of the pool size. The rate of synthesis is proportional to the incorporation into RNA and inversely proportional to the specific radioactivity of the precursor pool. The results of the incorporation experiments will be given below. The values obtained agreed very well with one another, irrespective of the media used. The UV absorption and phosphorus content per ,ug DNA, and the phosphorus content per A,,, unit were around 0.04 A,,, unit, 0.06 pg and 1.5 ,ug, respectively. These are about one-third, as compared with the values for whole embryos, which indicates loss of a remarkable amount of nucleoside derivatives during cell isolation and culture. From the present results it can be concluded that the pool size of the cells is fairly constant in all media. Thus, the specific radioactivity of the precursor pool is most likely to vary in proportion to the incorporation level into acid-soluble fractions.

Modification

of culture medium for Xenopus. I

243

Incorporation of 3H-uridine into acid-soluble fractions A set of the results obtained with Stearns medium is shown in fig. 1 a. Incorporation increased steeply and attained its maximum after 10 h. It then declined rather sharply to about an 80% level of the maximum in the next 14 h. This decrease was largely I

a 10

20

Fig. 2. Abscissa: time (hours); ordinate: cpm/embryo. o . 0.5 % albumin-containing medium (the original); l , 6.1 % a-globulin-containing medium; 0,-O.] % /3,y-globulin-containing medium; -----, acid-soluble; _. -, acid-insoluble. Incorporation of 3H-uridine into acid-soluble and insoluble fractions in a medium containing either 0.1 % or-globulin or 0.1 % jY,y-globulin. See legend for fig. 1. The specific radioactivity of 3H-uridine was one-tenth that in the experiments shown in fig. 1. Data are expressed in terms of cpm/ cells equivalent of an embryo.

1 b

Fig. I. Abscissa: time (hours); ordinate: cpm/embryo.

(a) 0.. . 0 , acid-soluble; o - 0 , acid-insoluble. Incorporation of 3H-uridine into acid-soluble and insoluble fractions in the original medium (containing 0.5 % albumin). (b) 0 . .rO , M NaCl-soluble; o -0, M NaCl-insoluble. Incorporation of 3Huridine into M NaCl-soluble and insoluble RNAs in the original medium (containing 0.5 % albumin). Embrvos were dissociated: the cells urenared were cultured and labeled for 4, IO and 24 h-in the medium containing 1 uCi/ml of 3H-uridine (5.0 Ciimmole). TCA-soluble and’ insoluble fractions‘were obtained. The RNA was also extracted and then fractionated into M NaCl-soluble and insoluble fractions. The radioactivity was measured. Data are expressed in terms of cpm per cells equivalent of an embryo.

due to exhaustion of the label outside the cells and the continuous flow into RNA, since the curve remained upward when a specific radioactivity of 3H-uridine was lowered (fig. 2). However, a possible impairment of the cellular activity cannot account for this, as incorporation of 3Huridine into RNA steadily increased throughout, as will be shown later. In the two modified media, in which a concentration of albumin was reduced to 0.1 y0 or was omitted, 3H-uridine uptake rose sharply with time for the first 10 h, then either declined slowly or reached a plateau. Thus, the curves were essentially the same as in the original medium, although the levels were only slightly elevated. Replacement of albumin with 0.1 “/o CCglobulin or 0.1% @,y-globulin changed the shape of the incorporation curves (fig. 2). The incorporation levels were at about 75-90x of controls at hour 10, but further increase occurred for the next 14 h, thus, at Exptl Cell Res 88 (1974)

244

Nakahashi and Yamana

Table 1. Incorporation of 3H-thymidine and 14C-protein hydrolysate into acid-insoluble material of Xenopus embryonic cells in various culture media Incorporation, cpm/cell equivalent

Medium containing 0.5 % albuminC 0.1 % albumin No albumin 0.1 % a-globulin 0.1 % P,y-globulin 3 % horse serum 3 % horse serum (dialysed)

W-protein 3H-thymidinea hydrolysateb Means ( %) Means ( %) 998 (loo) 697 (69.8) 562 (56.3) 1 131 (113) 1052 (105) 425 (42.6)

846 (100) 736 (87.0) 588 (69.5) 1067 (126) 867 (102) 442 (52.2)

259 (26.0)

294 (34.8)

Cells were obtained from blastulae and cultured for 24 h in the indicated media as described in Materials and Methods. The results are means of duplicate experiments and expressed in terms of cpm/cells eauivalent of an embrvo. 1-1 &i/ml (0.5 Ci/mmole). 0.05 ,&i/ml (1.99 mCi/mmole). ’ Stearns medium.

hour 24, attaining a level lo-25 % higher than that in the original medium. It was then assumed that the relative specific radioactivity was higher than that in the original. At the concentration used, p,y-globulin was more stimulatory than cc-globulin. When cultured in a medium containing 3 % horse serum, the cells showed a steeper rise in incorporation between hour 10 and 24. The radioactivity incorporated for 24 h in this medium amounted to approx. 1.5 times that in the original medium. Therefore, the specific radioactivity was about 1.5 times higher than in the latter. A medium containing 0.1% or 0.5 % protamine allowed no measurable incorporation into both acid-soluble and acid-insoluble fractions. After being cultured for 5 h in a 0.1 y0 protamin medium, the cells were no Exptl Cell Res 88 (1974)

longer viable. No aggregate formed: were signs of cell lysis.

there

Incorporation of 3H-uridine into RNA In the original medium the incorporation into acid-insoluble material or RNA began to rise with a short delay, and from then on proceeded linearly for the entire period of incubation (fig. 1a). The curves obtained appeared to be quite similar to those previously described [1, 21. With increasing time, the ratio of incorporation into RNA to that of acid-soluble fraction climbed steadily: at hour 24 about 40% of the total radioactivity was distributed in RNA. Certainly the major contribution to this was the enhanced incorporation into M NaClinsoluble RNA, which consisted largely of 18s and 28s ribosomal RNA (fig. 1b). The M NaCl-insoluble RNA : M NaCl-soluble RNA ratio began to rise at hour 10 and the increase lasted throughout the culture. This agrees with the previous findings that ribosomal RNA synthesis is activated and accelerated during this period of culture [ 1, 21. The RNA synthesized in isolated cells under the present conditions has been characterized [1, 21. It may be worth while noting here that the levels of incorporation into RNA varied much less widely than those of incorporation into acid-soluble fractions. The rate of 3H-uridine incorporation into RNA in a 0.1 % albumin medium was quite comparable to that in the original medium during 24 h. Furthermore, there was no change in the M NaCl-insoluble RNA: M NaCl-soluble RNA ratio. On the other hand, in a protein-free medium the cells incorporated label at a constant rate throughout incubation, but the rate was at most half that of controls. This is in remarkable contrast with the somewhat higher uptake into acidsoluble fractions in this medium. Judging from the relative specific radioactivity of the

Modification

of culture medium for Xenopus. I

245

Table 2. Aggregate formation by isolated Xenopus embryonic cells in various culture media Number of aggregates Size= (diameter in mm) Total Means (X)

0.2-0.4

Medium containing

Means ( %)”

0.40.6 Means ( %)”

> 0.6

0.5 96albumin* 0.1 % albumin No albumin 0.1 96 a-globulin 0.1 96 p, y-globulin 3 “b horse serum

201 (100) 129 (64) 131 (65) 208 (103) 240 (119) 264 (131)

182 (91) 118 (91) 119 (91) 190 (91) 206 (86) 245 (93)

18 (9) 11 (9) 11 (9) 18 (9) 31 (13) 17 (6)

1 0 1 0 3 2

Cells were obtained from blastulae and allowed to form aggregates in the indicated media as described in Materials and Methods. The results are means of 4 separate experiments. a Aggregates of less than 0.2 mm in diameter were neglected. ’ Stearns medium. ’ Y, of the total.

precursor pool, a 0.1 % albumin-containing medium supported RNA synthesis at a rate similar to that in the original medium, while the synthesis was reduced to a 50 % level in a protein-lacking medium. Any class of RNA, however, was not selectively inhibited, as the M NaCl-insoluble RNA : M NaCl-soluble RNA ratio did not change detectably. The cells showed essentially the same incorporation curves in a medium containing 0.1 y0 g-globulin or 0.1 % /3, y-globulin as in the original (fig. 2). This suggests that RNA was synthesizing at similar rates in both the modified and the original media. The relative rate of synthesis of M NaClinsoluble RNA to that of M NaCl-soluble RNA did not change. In the medium that was supplemented with 3 % horse serum, 3H-uridine was incorporated at a constant rate, but the rate was only about half that in Stearns medium. In addition, the relative specific radioactivity of the pool was sufficiently higher in the serum medium. Then, it is probable that the rate of RNA synthesis would be less than half that attained in the original medium. Again, this accompanied no change in the ratio of the RNA species synthesized.

Incorporation of 3H-thymidine and 14C-protein hydrolysate into acidinsoluble material The cells incorporated actively 3H-thymidine and 14C-protein hydrolysate into their acidinsoluble material in the original medium (table 1). Essentially the same trend was observed in previous works [l, 21. It has been demonstrated that in 0.1 % albumin and albumin-free media the incorporation of labels was greatly reduced, unlike that of 3H-uridine (table 1). Generally, 3H-thymidine incorporation appeared to be much more sensitive to protein deficiency than that of 14C-protein hydrolysate. Albumin is likely to have a favorable, either “toxicity-preventing” (or both), or “incorporation-promoting” effect on cells [5]. An a-globulin medium stimulated the incorporation into acid-insoluble material to 1lo-130 %, while /&y-globulin failed to do so (table 1). Although it has already been shown that cc-globulin promotes the growth of certain mammalian cells [5], there is no direct information about the rate of DNA and protein synthesis at present. However, if it can be assumed that the content of deoxynucleoside derivatives and amino acids, Exptl Cell Res 88 (1974)

246

Nakahashi and Yamana

like that of total nucleoside derivatives, was constant, irrespective of the different media used, the rate of DNA and protein synthesis in these modified media could be higher than that in the original medium. The cells showed incorporation at a rate that was about half that in the original medium, when cultured in a medium containing 3 % horse serum (table 1). In the present experiments, dialysed serum was less effective in stimulating the incorporation. What has already been found is that macromolecular component(s) in serum exert(s) a stimulatory effect on the multiplication of mammalian cells in culture [5]. The present observation seems thus to be somewhat incompatible. At present, however, no approach to this problem has been attempted. Aggregate formation The original medium allowed the cells to form about 200 aggregates during a 24 h culture under the present conditions (table 2). The aggregates were round or rod-shaped. Some of them had cilia all around their surface and were vigorously rotating. About 90 % of the aggregates had a diameter of 0.2-0.4 mm, the rest being in the range 0.4-0.6 mm. Cytological and cytochemical observations have revealed some characteristic cellular arrangement in them (unpublished). The ability to form aggregates was reduced to about 65 Y0in 0.1 % albumin or no albumin media. Their average size and appearance, however, did not differ from those of the aggregates in the original medium (table 2).

Exptl Cell Res 88 (1974)

Aggregates formed increased in number to about 120% in a p,y-globulin medium, but not in an cc-globulin medium (table 2). This is in contrast with the observation that cc-globulin, but not ,B,y-globulin, had a stimulatory effect on 3H-thymidine and 14C-protein hydrolysate incorporation. This may indicate that the aggregate-forming activity is not directly dependent on the synthesis activity. Changes in average size and appearance of aggregates were not observed. In a 3 % horse serum medium, an increased number of aggregates formed (about 150 “/‘), while their average size remained unchanged (table 2). Their appearance and shape were also similar to those in the original medium. This work was supported by the Ministry of Education. We thank Professor I. Kawakami for his suggestions and discussion.

REFERENCES 1. Yamana, K & Shiokawa, K, Proc Japan acad 42 (1966) 806. 2. Shiokawa, K & Yamana, K, Dev biol 16 (1967) 368. 3. Landesman, R & Gross, P R, Dev biol 18 (1968) 571. 4. Stearns. R N & Kostellow. A B. The chemical basis of development (ed W D ‘McElroy & B Glas) p. 448. Johns Hopkins Press, Baltimore, Md (1958). 5. Temin, H M, Pierson, jr, R W & Dulak, H C, Growth, nutrition, and metabolism of cells in culture (ed G H Rothblat & V J Cristofalo) vol. 1, p. 49. Academic Press, New York and London (1972). 6. Nieuwkoop, P D & Faber, J, Normal table of Xenoous laevis (Daudin). North-Holland Pub]., Amsierdam (1956). I. Abe, H & Yamana, K, Biochim biophys acta 240 (1970) 392. 8. Woodland, H R & Pestell, R Q W, Biochem j 127 (1972) 597. Received May 7, 1974